The present proposal will test the hypothesis that pathophysiologic changes in calcium handling (ie. uptake, efflux, buffering) play a major role in the initiation of renal epithelial cell injury during oxygen deprivation. Furthermore, this proposal will amplify our preliminary observations that calcium handling is different in anoxia and hypoxia in vitro, and that these differences do not remain fixed but become more pronounced as the time of oxygen deprivation increases. Specifically, the present proposal will investigate the role of K+ loss from cells and the resulting membrane depolarization induced by hypoxic or anoxic injury on the increased Ca2+ influx in rat proximal tubules (RPT) in vitro. In addition, the role of mitochondria in buffering this increased Ca2+ uptake will be determined by studies with ruthenium red (RR), lanthanum and FCCP addition followed by measurements of parameters of cell integrity (ie. adenine nucleotide levels, lactate dehydrogenase (LDH) release, mitochondrial respiration, and morphology). Activation of phospholipases in association with increases in cytosolic-free Ca2+ will be prevented by phospholipase inhibitors. The contribution of the Na+/Ca2+ exchanger causing the increase in cellular Na+ with oxygen deprivation will be examined by changing extracellular Na+ or Ca2+. The mechanism for the protective effects of glycine, ATP-MgCl2, adenosine and Mg2+ will also be evaluated. Special attention will be given to changes in Ca2+ uptake. The changes in Ca2+ kinetics (ie. increased uptake, decreased efflux, altered exchange rates and/or buffering capacity of mitochondria) will be evaluated in hypoxia and anoxia. The mechanisms of protective maneuvers (ie. impermeant solutes, acidosis, and calcium channel blockers) will be quantitated by morphologic and metabolic indices (ie. tissue electrolyte concentration, mitochondrial respiration, oxidative phosphorylation). The speed and degree of recovery upon reoxygenation of the above parameters will also be determined. These same indices will be evaluated during other protective maneuvers which reduce cellular depolarization. It is our hypothesis that maneuvers which prevent the changes in cellular electrolyte concentrations observed in oxygen deprivation studies will limit the increased cellular uptake of calcium and thereby lessen the severity of cell injury.